The E. coli non-ribosomal peptide synthetase (NRPS), a four-protein (EntE, B, D, F) six-module system is a paradigm for a bacterial "assembly-line" enzymes that produce molecules, such as antibiotics and metal-chelating siderophores. Our goal is to study the 142 kDa EntF component consisting of a condensation domain (C,49 kDa), an adenylase (A, 59 kDa) a peptidyl carrier or thiolation module (T, 9 kDa), and a thioesterase (TE, 27 kDa). EntF cooperates with EntB which consists of an A and a T domain. We plan to solve structures of individual domains and several di-domain constructs or even larger portions using modern NMR methods. Of particular interest will be to elucidate the orientation of the individual domains relative to each other, which will be pursued by measuring residual dipolar couplings and utilizing paramagnetic broadening of NMR signals by strategically induced spin labels. The relative orientation of the domains is of crucial importance for understanding the mechanism of how the fragments of the growing peptide chain are brought together. The research will be pursued with four specific aims. Aim 1 is to establish expression and isotope labeling of individual domains of EntF and EntB, as well as di- tri- and tetra-domain constructs and to explore the feasibility of NMR spectroscopy of the constructs. In this aim we will include segmental labeling using inteins or related technologies. Aim 2 is to perform resonance assignments starting with the single domains and proceeding to multi-domain constructs of increasing complexity. Aim 3 is to solve structures of individual domains an larger fragments. Aim 4 is to elucidate the orientations of the domains to each other and relative to the individual active sites.